The Interaction between Canine Semen Bacteria and Semen Quality Parameters.

Assessing canine semen quality helps to detect infertility in males, but identifying factors that influence canine semen quality is a complicated task. The objective of this study was the assessment of the potential influence of bacteria found in canine semen samples on the characteristics of dogs' semen. In this study, semen samples were collected manually from 30 dogs and subjected to a comprehensive examination. The results of sperm motility, concentration, viability, and morphology were statistically analysed in relation to the number of bacteria in the semen (CFUs/mL) and the seminal microbiota. Samples with an increased bacterial count per millilitre were associated with lower-quality sperm motility (p < 0.05). The most frequently isolated bacterial genera from the analysed semen samples were Staphylococcus spp. (26.0%), Corynebacterium spp. (17.8%), and Streptococcus spp. (16.4%). The presence of ß-haemolytic Escherichia coli bacteria was linked to suboptimal semen samples, characterised by significantly reduced semen viability and a lower proportion of morphologically normal spermatozoa (p < 0.05). Corynebacterium spp. was associated with reduced bacterial load and superior semen quality (p < 0.01). These findings highlight the importance of bacterial cell counts and microbiota diversity in relation to various factors influencing canine semen quality, providing a more comprehensive understanding of canine reproductive well-being.

Microbial Composition of Extracted Dental Alveoli in Dogs with Advanced Periodontitis.

Periodontitis is a serious gum infection that damages the soft tissue and destroys the bone supporting the teeth. The aim of the study was to investigate the microbiota using traditional microbiology plating and metagenomic sequencing of extracted tooth alveoli in dogs with severe periodontitis. Isolation of culturable microorganisms was performed as part of bacteriological testing to provide bacteriological diagnosis to veterinary surgeons. Metagenomic sequencing was performed using shotgun sequencing on the Illumina HiSeq system platform. The most prevalent species at sites of periodontal infection detected by metagenomic sequencing were Porphyromonas gulae, Prevotella spp., Tannerella forsythia, Porphyromonas crevioricanis, Porphyromonas cangingivalis, and Bacteroides heparinolyticus. Pasteurella, Streptococcus, and Neisseria were the most frequently isolated culturable bacteria from infected sites detected by traditional microbiologic methods. Metagenomic data revealed that these three genera accounted for only 1.6% of all microbiota at the sites of infection. Antimicrobial resistance patterns of the isolated bacteria included resistance to ampicillin, doxycycline, sulfamethoxazole-trimethoprim, ciprofloxacin, colistin, cefotaxime, and chloramphenicol. Antimicrobial-resistant genes detected using shotgun sequencing also showed resistance to aminoglycosides and macrolides. Dogs with periodontal infections carry bacteria that can cause bite infections in humans as well as multi-resistant isolates. Therefore, treatment and prophylaxis or periodontal disease of dogs is important from a One Health perspective.

A Study on the Stability and Antimicrobial Efficacy of a Newly Modeled Teat Dip Solution Containing Chlorhexidine.

Despite much focus on mastitis as an endemic disease, clinical and subclinical mastitis remains an important problem for many herds. Reducing the usage of antibiotics for mastitis treatment allows the risks to be minimized related to the development of antimicrobial resistance and the excretion of antibiotics into the environment. The aim of the study was to determine the physico-chemical properties, stability and antimicrobial effect of a newly formulated biocide for post-milking udder hygiene containing a thickener made from hydroxypropyl guar gum, an antiseptic chlorhexidine digluconate and teat skin-friendly components including glycerol, Mentha Arvensis herbal oil and Aesculus hippocastanum extract. Hydroxypropyl guar gum was used as a thickener to provide the physical parameters and to retain the viscosity at 1438 mPa.s. The physical and chemical properties of the product, including the 12-month stability, were tested in long-term and accelerated stability studies. The product was effective against the primary mastitis pathogens, including Staphylococcus aureus, Streptococcus uberis, Escherichia coli, Candida albicans and Aspergillus niger.

Serological Variety and Antimicrobial Resistance in Salmonella Isolated from Reptiles.

Salmonella enterica is one of the best adapted bacterial pathogens causing infections in a wide variety of vertebrate species. The aim of this study was to investigate the prevalence of Salmonella in different reptile species and to evaluate their serological variety and patterns of antimicrobial resistance. In total, 97 samples from 25 wild and domesticated reptile species were investigated in Lithuania. Serological variety, as well as phenotypical and genotypical resistance to antimicrobials, were investigated. Fifty isolates of Salmonella were obtained from the ninety-seven tested samples (51.5%; 95% CI 41.2−61.2). A significantly higher prevalence of Salmonella was detected in domesticated individuals (61.3%; 95% CI 50.0−71.5) compared with wild ones (18.2%; 95% CI 7.3−38.5). All isolates belonged to a single species, Salmonella enterica. Results demonstrated that reptiles carry a large variety of Salmonella serovars. Thirty-four isolates (68%) of Salmonella were resistant to at least one antimicrobial drug. The most frequent resistance of the isolates was to streptomycin (26%), cefoxitin, gentamicin, tetracycline and chloramphenicol (16%). Genes encoding resistance to tetracyclines, aminoglycosides, sulphonamides and trimethoprim were detected. No integrons that are associated with horizontal gene transfer were found. Data obtained provided knowledge about the adaptation of Salmonella in reptiles. Healthy individuals, irrespective of their origin, often carry Salmonella, including multi-resistant strains. Due to its large serological diversity, zoonotic potential and antimicrobial resistance, Salmonella in reptiles poses a risk to other animals and humans.

CTX-M-producing Escherichia coli strains: resistance to temocillin, fosfomycin, nitrofurantoin and biofilm formation.

Aim: ESBL-producing and bacterial biofilms-forming Escherichia coli are associated with antimicrobial treatment failure. This study aimed to investigate the phenotypic resistance mechanisms of CTX-M E. coli against old antibiotics - cell wall synthesis inhibitors temocillin, nitrofurantoin and fosfomycin. Materials & Methods: Susceptibility to old antibiotics testing was performed using disk diffusion method, biofilm formation was evaluated spectrophotometrically, and PCR was used for the determination of CTX-M type. Results & conclusion: Temocillin was active against nearly 93%, nitrofurantoin and fosfomycin, respectively, 91.7% and 98.6% of tested E. coli. Thus, it demonstrated to be a good alternative therapeutic option against ESBL infections. Bacteria resistant to old antibiotics had CTX-M-15 or CTX-M-15, TEM-1 and OXA-1 combinations. No significant association was found between CTX-M E. coli resistance to temocillin, nitrofurantoin and fosfomycin; however, the level of biofilm formation was found as not affected by the type of CTX-M ß-lactamases.

Microbiome and Antimicrobial Resistance Genes in Microbiota of Cloacal Samples from European Herring Gulls (Larus Argentatus).

INTRODUCTION: The aim of the study was to determine microbiota in the cloacal samples of European herring gulls (Larus argentatus) and to compare a variety of genes encoding antimicrobial resistance in cultivable and non-cultivable bacteria. MATERIAL AND METHODS: Cloacal samples from European herring gulls were collected from a Kaunas city dump. Cultivable microbiota were isolated, their microbial susceptibility was tested, and genes encoding antimicrobial resistance were detected. Additionally, a metagenomic study was performed using Next-Generation Sequencing (NGS). RESULTS: In total, 697 different operational taxonomic units at genus level were detected; however, only 63 taxonomic units were detected at the amount of ≥0.1% of the total number of DNA copies. Catellicoccus marimammalium was found to have the highest prevalence. The bacterial amount of other genera was up to 5% with the most highly prevalent being Psychrobacter (4.7%), Helicobacter(4.5%), unclassified Enterococcaceae (3.2%), Pseudomonas (2.9%), and Brachyspira (2.6%). CONCLUSIONS: C. marimammalium are predominant microbiota in the cloacal samples of Larus argentatus. This species of gulls is a reservoir of bacteria carrying a wide-spectrum of genes encoding antimicrobial resistance. The same genes were detected in both cultivable microbiota and in the total DNA of the samples.

DETERMINATION OF ANTIMICROBIAL ACTIVITY OF CHLORHEXIDINE GEL.

Many researchers have advocated in recent times that antiseptic use in healing wounds should be discouraged. Antiseptics have been found to retard healing of wounds. Poloxamer 407 shows thermoreversible properties, which are of the utmost interest in optimizing drug formulation (fluid state at room temperature facilitating administration and gel state above sol-gel transition temperature, at body temperature, promoting prolonged release of pharmacological agents). Chlorhexidine, a commonly used antiseptic, is known to be less toxic on granulation cells. Acting as an antiseptic, it is an effective bactericidal agent against the most categories of microbes, including bacteria, yeast, and viruses. Objective of this study was to evaluate antimicrobial activ- ity of chlorhexidine containing poloxamer gel to Gram-positive and Gram-negative bacteria in vitro. Chlorhexidine gels and chlorhexidine aqueous solutions have different antibacterial activity to S. amis, E.faecalis, E. coli and P. aemginosa strains in vitro. It depends on concentration and dosage form of antiseptic. Study results confirmed that antimicrobial activity of gel depends on active ingredient concentration in antiseptic. The best inhibition effect for both of reference and wild-type bacteria was obtained for 1% chlorhexidine gel. Summarizing the results and assessing the characteristics of the gel ingredients, it can be suggested using chlorhexidine gels in veterinary medicine.

Prevalence, species distribution and antimicrobial resistance patterns of methicillin-resistant staphylococci in Lithuanian pet animals.

BACKGROUND: The bacterial genus Staphylococcus consists of many species that causes infections in pet animals. Antimicrobial resistant staphylococci cause infections that are difficult to treat and they are important from the point of one health perspective. The aim of this study was to determine the prevalence of methicillin-resistant Staphylococcus (MRS) species, including methicillin-resistant S. aureus (MRSA) in diseased pet animals (Group A) and kennel dogs (Group B) in Lithuania and to characterize the isolates according to their antimicrobial resistance. RESULTS: Twenty-one MRS isolates were obtained from 395 clinical samples (5.3 %; CI 95 % 3.5-8.0) of Group A animals. Sixteen, four and one isolates were from dogs, cats and a pet rabbit, respectively. The mecA gene was present in 20 isolates, whereas one isolate was positive for the mecC gene. Twenty-one MRS isolates (20.0 %; CI 95 % 13.5-28.6) were obtained from the vagina of female dogs (n = 105) (Group B). All isolates carried the mecA gene. Twelve MRS species were isolated of which S. pseudintermedius was the most common (18/42) followed by S. haemolyticus (8/42) and S. lentus (4/42). MRSA was not found. All MRS strains were susceptible to vancomycin, linezolid, daptomycin and quinupristin/dalfopristin. Resistance to tetracycline (16/21), clindamycin (15/21) and erythromycin (14/21) was the most common types of resistance in Group A animals. Three isolates also demonstrated resistance to rifampin. Resistance toward gentamicin (16/21), ciprofloxacin (15/21), macrolides (15/21) and tetracycline (12/21) was the most common in kennel dogs (Group B). The most common genes encoding resistance to antimicrobials (excluding beta-lactams) in isolates from Group A pets were tetK (21/42), aph(3')-IIIa (11/42) and aac(6')-Ie-aph(2'')-Ia (9/42). CONCLUSIONS: A wide range of MRS species were found in pet animals in Lithuania. MRSA was not found.

High-yield production of a functional bacteriophage lysin with antipneumococcal activity using a plant virus-based expression system.

Streptococcus pneumoniae is the causative agent of several serious infectious diseases. It is becoming increasingly antibiotic resistant worldwide, and thus new antimicrobials are needed. One alternative to antibiotics may be the use of peptidoglycan hydrolases, the bacteriophage lytic enzymes. In this study, we demonstrated high level expression of the S. pneumoniae bacteriophage lysin Pal in Nicotiana benthamiana - TMV (Tobacco Mosaic Virus) transient expression system. The protein was purified to homogeneity and tested for streptococci killing activity in vitro and in vivo. In vitro, Pal was able to lyse three tested S. pneumoniae strains: NCTC12695, NCTC12977 and NCTC11888. The treatment of BALB/c mice with 100 µg, 200 µg and 400 µg of Pal 1h post-challenge with double lethal dose of S. pneumoniae NCTC12695 strain showed a clear dose response and protected from lethal sepsis 30%, 40% and 50% of mice, respectively. The improved mice survival correlated with decreased blood bacterial titers. In conclusion, these results suggest that plant-expressed bacteriophage lysins may have potential use as antimicrobial agents.

Prevalence of methicillin-resistant Staphylococcus haemolyticus in companion animals: a cross-sectional study.

BACKGROUND: Among coagulase-negative staphylococci, Staphylococcus haemolyticus is the second most frequently isolated species from human blood cultures and has the highest level of antimicrobial resistance. This species has zoonotic character and is prevalent both in humans and animals. Recent studies have indicated that methicillin-resistant S. haemolyticus (MRSH) is one of the most frequent isolated Staphylococcus species among neonates in intensive care units. The aim of this study was to determine the presence of MRSH in different groups of companion animals and to characterize isolates according their antimicrobial resistance. METHODS: Samples (n = 754) were collected from healthy and diseased dogs and cats, female dogs in pure-breed kennels, healthy horses, and kennel owners. Classical microbiological tests along with molecular testing including PCR and 16S rRNA sequencing were performed to identify MRSH. Clonality of the isolates was assessed by Pulsed Field Gel Electrophoresis using the SmaI restriction enzyme. Antimicrobial susceptibility testing was performed using the broth micro-dilution method. Detection of genes encoding antimicrobial resistance was performed by PCR. Statistical analysis was performed using the R Project of Statistical Computing, "R 1.8.1" package. RESULTS: From a total of 754 samples tested, 12 MRSH isolates were obtained. No MRSH were found in horses and cats. Eleven isolates were obtained from dogs and one from a kennel owner. Ten of the dog isolates were detected in pure-breed kennels. The isolates demonstrated the same clonality only within separate kennels.The most frequent resistances of MRSH isolates was demonstrated to benzylpenicillin (91.7%), erythromycin (91.7%), gentamicin (75.0%), tetracycline (66.7%), fluoroquinolones (41.7%) and co-trimoxazole (41.7%). One isolate was resistant to streptogramins. All isolates were susceptible to daptomycin, rifampin, linezolid and vancomycin. The clone isolated from the kennel owner and one of the dogs was resistant to beta-lactams, macrolides, gentamicin and tetracycline. CONCLUSIONS: Pure-breed kennels keeping 6 or more females were determined to be a risk factor for the presence of MRSH strains. MRSH isolated from companion animals were frequently resistant to some classes of critically important antimicrobials, although they remain susceptible to antibiotics used exclusively in human medicine.

Transferable class 1 and 2 integrons in Escherichia coli and Salmonella enterica isolates of human and animal origin in Lithuania.

Antibiotic-resistant Escherichia coli (n = 191) and Salmonella enterica (n = 87) isolates of human and animal origin obtained in Lithuania during 2005-2008 were characterized for the presence and diversity of class 1 and 2 integrons. E. coli isolates were obtained from patients with urinary tract infections (UTIs) (n = 59) and both healthy and diseased farm animals, including poultry (n = 54), swine (n = 35), and cattle (n = 43). Isolates of non-typhoidal S. enterica were recovered from salmonellosis patients (n = 37) and healthy animals, including poultry (n = 31) and swine (n = 19). The presence of integrons, their gene cassette structure, and genome location were investigated by polymerase chain reaction, restriction fragment-length polymorphism, DNA sequencing, Southern blot hybridization, and conjugation experiments. Forty percent of the E. coli and 11% of the S. enterica isolates carried class 1 integrons, whereas class 2 integrons were found in E. coli isolates (9%) only. The incidence of integrons in human UTIs and cattle isolates was most frequent (p < 0.01). A total of 23 different gene cassettes within 15 different variable regions were observed. Seven different integron types, all of them transferable by conjugation, were common for isolates from human infections and for one or more groups of animal isolates. The most prevalent integron types contained arrays dfrA1-aadA1 (36%), dfrA17-aadA5 (23%), and dfrA1-sat1-aadA1 (78%). Two E. coli isolates from humans with UTIs harbored class 1 integron on conjugative plasmid with the novel array type of 4800 bp/dfrA17-aadA5Δ-IS26-ΔintI1-aadB-aadA1-cmlA residing on the Tn21-like transposon. Three S. enterica isolates from swine contained class 1 integron with the newly observed array type of 1800 bp/aadA7-aadA7. Integrons of 10 different types of both classes were located on transferable plasmids in E. coli and S. enterica. Our study demonstrated the existence of a considerable and common pool of transferable integrons in E. coli and S. enterica present in clinical and livestock environment in Lithuania.